Burkitt lymphoma carries multiple oncogenic drivers yet arises predominantly in children, whose normal cells harbour few age-related mutations. To investigate this paradox, we seek to define the sequence and timing of mutational drivers underlying Burkitt lymphoma development. Here, we analyse single-cell whole-genome sequencing data of 250 paired normal and malignant B-cells from Burkitt lymphoma patients and integrate this data with 21 bulk WGS samples and an existing dataset of 157 clonally expanded B-cells from healthy individuals. Phylogenetic reconstruction reveals an accelerated accumulation of mutations following the emergence of the most recent common ancestor, leading to the early establishment of extensive genetic intratumoural heterogeneity. We further provide evidence that convergent evolution shapes this diversity at both the point mutation- and copy number variation-levels. This in-depth characterisation further establishes Burkitt lymphoma as a paradigmatic model of tumorigenesis with implications for therapeutic intervention.

Outcomes for pediatric non-Hodgkin lymphoma (NHL) have improved markedly over the past decade; however, reducing therapy-related toxicity while maintaining excellent survival remains a major challenge. This narrative review, conducted using a PCC (Population–Concept–Context) framework, summarizes current evidence on large B-cell lymphoma with IRF4-rearrangement (LBCL-IRF4⁺) in children, adolescents, and young adults (CAYA) below 30 years of age. Focusing on clinical presentation, pathological and molecular features, treatment strategies, and outcomes, based on literature searches in PubMed, Embase, and Web of Science performed in November 2025. LBCL-IRF4⁺ is a rare subtype, and evidence among these age groups is largely derived from small retrospective series. Available data indicate that patients with low-stage disease and/or follicular growth patterns may be candidates for treatment de-escalation, while those with advanced-stage or atypical presentations should be evaluated for underlying immunologic predispositions. This review underscores the importance of including LBCL-IRF4⁺ in future clinical trials to optimize therapy intensity and explore opportunities for safe de-escalation in selected patient groups.

In contrast to RNA expression, which captures the current state of a cell, DNA is the only biomolecule in a cell that is not constantly replaced but copied and passed on during cell divisions. As a result, mutations that occur in the DNA code are fixed and gradually accumulate forming an archive of the life history of cells. DNA from pediatric cancers usually do not have high mutational loads. However, this is not the case with pediatric lymphomas which tend to harbor many mutations. This makes pediatric lymphomas an ideal model disease to study mutation accumulation. Here, we have applied state-of-the-art single-cell whole genome sequencing on pediatric Burkitt, T-lymphoblastic and Hodgkin lymphoma using the Primary Template-directed Amplification technique. Using this method, we have compared the genomes of malignant and non-malignant cells from the same tumors. We have also included patients in our cohort which have multiple-site involvement to investigate clonal dynamics across tissues. Using in-depth mutational analyses, we have generated detailed phylogenetic trees of each patient, identified processes unique to tumorigenesis and timed the occurrence of driver mutations and mutagenic processes. This type of analysis has not been applied to lymphoma before. Outcome of lymphoma in children is excellent (>90% survival), whereas survival in adults is much lower (~50%), because children can be treated with higher doses of chemotherapy. Consequently, treatment is extremely toxic, and relapses are mostly lethal. We envision that our research will contribute to improved diagnostics and ultimately the development of more targeted, less toxic treatment and a strongly improved quality of life.

Twenty percent of children with T-cell lymphoblastic lymphoma (T-LBL) will relapse and have an extremely poor outcome. Currently, we can identify a genetically low-risk subgroup in pediatric T-LBL, yet these high-risk patients who need intensified or alternative treatment options remain undetected. Therefore, there is an urgent need to recognize these high-risk T-LBL patients through identification of molecular characteristics and biomarkers. By using RNA sequencing which was performed in 29/49 T-LBL patients who were diagnosed in the Princess Maxima Center for Pediatric Oncology between 2018 and 2023, we discovered a previously unknown high-risk biological subgroup of children with T-LBL. This subgroup is characterized by NOTCH1 gene fusions, found in 21% of our T-LBL cohort (6/29). All patients presented with a large mediastinal mass, pleural/pericardial effusions, and absence of blasts in the bone marrow, blood, and central nervous system. Blood CCL17 (C-C Motif Chemokine Ligand 17, TARC) levels were measured at diagnosis in 26/29 patients, and all six patients with NOTCH1 gene fusions patients exclusively expressed highly elevated blood CCL17 levels, defining a novel and previously not known clinically relevant biomarker for T-cell lymphoblastic lymphoma. Four out of these six patients relapsed during therapy, a fifth developed a therapy-related acute myeloid leukemia during maintenance therapy. These data indicate that T-LBL patients with a NOTCH1 fusion have a high risk of relapse which can be easily identified using a blood CCL17 screening at diagnosis. Further molecular characterization through NOTCH1 gene fusion analysis offers these patients the opportunity for treatment intensification or new treatment strategies.

AbstractPediatric classic Hodgkin lymphoma (cHL) patients have a high survival rate but suffer from severe long‐term side effects induced by chemo‐ and radiotherapy. cHL tumors are characterized by the low fraction (0.1%–10%) of malignant Hodgkin and Reed–Sternberg (HRS) cells in the tumor. The HRS cells depend on the surrounding immune cells for survival and growth. This dependence is leveraged by current treatments that target the PD‐1/PD‐L1 axis in cHL tumors. The development of more targeted therapies that are specific for the tumor and are therefore less toxic for healthy tissue compared with conventional chemotherapy could improve the quality of life of pediatric cHL survivors. Here, we applied single‐cell RNA sequencing (scRNA‐seq) on isolated HRS cells and the immune cells from the same cHL tumors. Besides TNFRSF8 (CD30), we identified other genes of cell surface proteins that are consistently overexpressed in HRS cells, such as NRXN3 and LRP8, which can potentially be used as alternative targets for antibody–drug conjugates or CAR T cells. Finally, we identified potential interactions by which HRS cells inhibit T cells, among which are the galectin‐1/CD69 and HLA‐II/LAG3 interactions. RNAscope was used to validate the enrichment of CD69 and LAG3 expression on T cells near HRS cells and indicated large variability of the interaction strength with the corresponding ligands between patients and between tumor tissue regions. In conclusion, this study identifies new potential therapeutic targets for cHL and highlights the importance of studying heterogeneity when identifying therapy targets, specifically those that target tumor‐immune cell interactions.

Histiocytic disorders are rare hematologic neoplasms characterized by a notable dependence on mitogen-activated protein kinase signaling. With conventional therapies, a substantial proportion of histiocytosis patients experiences disease progression. Targeted therapy is an emerging treatment option, with the capability to induce robust responses. Yet, the number of reported patients remains limited. Here, we describe 40 patients with histiocytic neoplasms who were treated with targeted therapy in 7 hospitals in the Netherlands and Belgium. The cohort comprised 6 (15%) children and 34 (85%) adults with diverse histiocytoses, including Langerhans cell histiocytosis (LCH; n=12), Erdheim-Chester disease (n=14), central nervous system xanthogranuloma (n=2), Rosai-Dorfman disease (n=3), histiocytic sarcoma (n=2), ALK-positive histiocytosis (n=1), and mixed/unclassifiable histiocytosis (n=6). Five patients were included in a clinical trial; 35/40 (88%) received BRAF/MEK inhibitors outside of trials. Among these 35 patients with available follow-up data, median time on targeted treatment was 1.9 years (range: 0.1 – 5.8 years). Complete or partial responses were observed in 25/27 (93%) patients treated for multisystemic and/or solid lesions and 2/8 (25%) patients treated for neurodegenerative LCH. Responses were generally durable, although 10 patients lost response after dose reduction or therapy interruption. Responses were recaptured in 9/10 cases. Two patients developed new or progressive neurodegenerative lesions: 1 during and 1 after vemurafenib therapy. At last follow-up, 8/35 (23%) patients had stopped targeted therapy because of toxicity – all of whom were adults. This study corroborates the favorable outcomes of BRAF/MEK inhibition in histiocytosis patients described previously. However, it also highlights limitations and calls for prospective studies.

Background Immunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab) antibody was reformatted into the IgA1 isotype, which abolishes neuropathic pain and induces efficient neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) via activation of the Fc alpha receptor (FcαRI/CD89). Methods To generate an antibody suitable for clinical application, we engineered an IgA molecule (named IgA3.0 ch14.18) with increased stability, mutated glycosylation sites and substituted free (reactive) cysteines. The following mutations were introduced: N45.2G and P124R (CH1 domain), C92S, N120T, I121L and T122S (CH2 domain) and a deletion of the tail piece P131-Y148 (CH3 domain). IgA3.0 ch14.18 was evaluated in binding assays and in ADCC and antibody-dependent cellular phagocytosis (ADCP) assays with human, neuroblastoma patient and non-human primate effector cells. We performed mass spectrometry analysis of N-glycans and evaluated the impact of altered glycosylation in IgA3.0 ch14.18 on antibody half-life by performing pharmacokinetic (PK) studies in mice injected intravenously with 5 mg/kg antibody solution. A dose escalation study was performed to determine in vivo efficacy of IgA3.0 ch14.18 in an intraperitoneal mouse model using 9464D-GD2 neuroblastoma cells as well as in a subcutaneous human xenograft model using IMR32 neuroblastoma cells. Binding assays and PK studies were compared with one-way analysis of variance (ANOVA), ADCC and ADCP assays and in vivo tumor outgrowth with two-way ANOVA followed by Tukey’s post-hoc test. Results ADCC and ADCP assays showed that particularly neutrophils and macrophages from healthy donors, non-human primates and patients with neuroblastoma are able to kill neuroblastoma tumor cells efficiently with IgA3.0 ch14.18. IgA3.0 ch14.18 contains a more favorable glycosylation pattern, corresponding to an increased antibody half-life in mice compared with IgA1 and IgA2. Furthermore, IgA3.0 ch14.18 penetrates neuroblastoma tumors in vivo and halts tumor outgrowth in both 9464D-GD2 and IMR32 long-term tumor models. Conclusions IgA3.0 ch14.18 is a promising new therapy for neuroblastoma, showing (1) increased half-life compared to natural IgA antibodies, (2) increased protein stability enabling effortless production and purification, (3) potent CD89-mediated tumor killing in vitro by healthy subjects and patients with neuroblastoma and (4) antitumor efficacy in long-term mouse neuroblastoma models.

While cervical lymphadenopathy is common in children, a decision model for detecting high-grade lymphoma is lacking. Previously reported individual lymphoma-predicting factors and multivariate models were not sufficiently discriminative for clinical application. To develop a diagnostic scoring tool, we collected data from all children with cervical lymphadenopathy referred to our national pediatric oncology center within 30 months (n = 182). Thirty-nine putative lymphoma-predictive factors were investigated. The outcome groups were classical Hodgkin lymphoma (cHL), nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), non-Hodgkin lymphoma (NHL), other malignancies, and a benign group. We integrated the best univariate predicting factors into a multivariate, machine learning model. Logistic regression allocated each variable a weighing factor. The model was tested in a different patient cohort (n = 60). We report a 12-factor diagnostic model with a sensitivity of 95% (95% CI 89-98%) and a specificity of 88% (95% CI 77-94%) for detecting cHL and NHL. Our 12-factor diagnostic scoring model is highly sensitive and specific in detecting high-grade lymphomas in children with cervical lymphadenopathy. It may enable fast referral to a pediatric oncologist in patients with high-grade lymphoma and may reduce the number of referrals and unnecessary invasive procedures in children with benign lymphadenopathy.

Since mice do not express a homologue of the human Fc alpha receptor (FcαRI or CD89), a transgenic mouse model was generated in four different backgrounds (C57BL/6, BALB/c, SCID and NXG) expressing the FcαRI under the endogenous human promoter. In this study, we describe previously unknown characteristics of this model, such as the integration site of the FCAR gene, the CD89 expression pattern in healthy male and female mice and in tumor-bearing mice, expression of myeloid activation markers and FcγRs and IgA/CD89-mediated tumor killing capacity. In all mouse strains, CD89 expression is highest in neutrophils, intermediate on other myeloid cells such as eosinophils and DC subsets and inducible on, among others, monocytes, macrophages and Kupffer cells. CD89 expression levels are highest in BALB/c and SCID, lower in C57BL/6 and lowest in NXG mice. Additionally, CD89 expression on myeloid cells is increased in tumor-bearing mice across all mouse strains. Using Targeted Locus Amplification, we determined that the hCD89 transgene has integrated in chromosome 4. Furthermore, we established that wildtype and hCD89 transgenic mice have a similar composition and phenotype of immune cells. Finally, IgA-mediated killing of tumor cells is most potent with neutrophils from BALB/c and C57BL/6 and less with neutrophils from SCID and NXG mice. However, when effector cells from whole blood are used, SCID and BALB/c are most efficient, since these strains have a much higher number of neutrophils. Overall, hCD89 transgenic mice provide a very powerful model to test the efficacy of IgA immunotherapy against infectious diseases and cancer.

Introduction: Burkitt lymphoma (BL) is a rare but highly aggressive B-cell non-Hodgkin lymphoma. BL exhibits a characteristic immunophenotype that is positive for pan-B cell markers and CD10. The genetic hallmark of BL is the translocation of the MYC oncogene under the regulation of an immunoglobulin (IG) heavy or light chain enhancer, resulting in MYC protein overexpression. Notably, MYC translocation by itself is not sufficient for BL oncogenesis and a variety of cancer genes are recurrently mutated. BL can be divided epidemiologically but also, more recently, genetically based on driver mutations including DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Despite high survival rates, pediatric BL patients suffer from long-term side effects and relapses are usually fatal. In order to develop more targeted and less toxic therapies, a better understanding is needed of the ethology of the disease. Therefore, our aim is to characterise the cell-of-origin (COO) and dissect the life history of BL subtypes, by pinpointing when during tumorigenesis somatic mutations play a role and by identifying the rate-limiting steps of malignant transformation. Method: We collected single-cell suspensions of BL patient samples, including lymph node biopsies, bone marrow aspirates, as well as ascites and pleural fluid. To perform flow cytometry, single cells were stained with DAPI and a panel of antibodies: CD10/CD45/CD19 or CD10/CD3/CD20. Live B cells were separated with the DAPI-CD45+ CD19+ or DAPI-CD3-CD20+ phenotype. Subsequently, CD10+ B cells and CD10- B cells were sorted in 96-well plates (Figure 1). To confirm the presence or absence of the IG::MYC translocation a PCR was perfomed using patient-specific primers that flank the translocation locus. Whole genome amplification was carried out on the DNA of single cells using the Primary Template-directed Amplification (PTA) technique that was subsequently whole genome sequenced. Results: From a bone marrow sample of a 4-year-old female BL patient we found that 1% of immune cells were CD10+ B cells, while 17% of B cells were negative for the CD10 marker (Figure 2A). In the lymph node sample from the same patient, we found that 87% of B cells were CD10+ and 9% were CD10- (Figure 2B). PCR confirmed the MYC translocation in 2/6 CD10+ and 0/6 CD10- bone marrow-derived B cells (Figure 3). Keywords: Genomics, Epigenomics, and Other -Omics, Non-Hodgkin (Pediatric, Adolescent, and Young Adult), Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

IntroductionClassical Hodgkin lymphoma (cHL) is the most common pediatric lymphoma. Approximately 10% of patients develop refractory or recurrent disease. These patients are treated with intensive chemotherapy followed by consolidation with radiotherapy or high-dose chemotherapy and autologous stem cell reinfusion. Although this treatment is effective, it comes at the cost of severe long-term adverse events, such as reduced fertility and an increased risk of secondary cancers. Recently, promising results of inducing remission with the immune checkpoint inhibitor nivolumab (targeting PD-1) and the anti-CD30 antibody-drug conjugate Brentuximab vedotin (BV) +/- bendamustine were published.MethodsHere we describe a cohort of 10 relapsed and refractory pediatric cHL patients treated with nivolumab + BV +/- bendamustine to induce remission prior to consolidation with standard treatment.Results and discussionAll patients achieved complete remission prior to consolidation treatment and are in ongoing complete remission with a median follow-up of 25 months (range: 12 to 42 months) after end-of-treatment. Only one adverse event of CTCAE grade 3 or higher due to nivolumab + BV was identified. Based on these results we conclude that immunotherapy with nivolumab + BV +/- bendamustine is an effective and safe treatment to induce remission in pediatric R/R cHL patients prior to standard consolidation treatment. We propose to evaluate this treatment further to study putative long-term toxicity and the possibility to reduce the intensity of consolidation treatment.

The study was conducted to assess the feasibility of integrating state-of-the-art sequencing techniques and flow cytometry into diagnostic workup of pediatric lymphoma. RNA sequencing (RNAseq), whole exome sequencing, and flow cytometry were implemented into routine diagnostic workup of pediatric biopsies with lymphoma in the differential diagnosis. Within 1 year, biopsies from 110 children (122 specimens) were analyzed because of suspected malignant lymphoma. The experience with a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies is reported. Flow cytometry was performed with fresh tissue in 83% (102/122) of specimens and allowed rapid diagnosis of T-cell and B-cell non-Hodgkin lymphomas. RNAseq was performed in all non-Hodgkin lymphoma biopsies and 42% (19/45) of Hodgkin lymphoma samples. RNAseq detected all but one of the translocations found by fluorescence in situ hybridization and PCR. RNAseq and whole exome sequencing identified additional genetic abnormalities not detected by conventional approaches. Finally, 3 cases are highlighted to exemplify how synergy between different diagnostic techniques and specialists can be achieved. This study demonstrates the feasibility and discusses the added value of integrating modern sequencing techniques and flow cytometry into a workflow for routine diagnostic workup of lymphoma. The inclusion of RNA and DNA sequencing not only supports diagnostics but also will lay the ground for the development of novel research-based treatment strategies for pediatric lymphoma patients.

The European Intergroup for Childhood Non-Hodgkin Lymphoma (EICNHL) was established 25 years ago with the goal to facilitate clinical trials and research collaborations in the field both within Europe and worldwide. Since its inception, much progress has been made whereby major improvements in outcomes have been achieved. In this Review, we describe the different diagnostic entities of non-Hodgkin lymphoma in children and young adults describing key features of each entity and outlining clinical achievements made in the context of the EICNHL framework. Furthermore, we provide an overview of advances in biopathology with an emphasis on the role of biological studies and how they have shaped available treatments. Finally, for each entity, we describe future goals, upcoming clinical trials, and highlight areas of research that require our focus going forward.

Lymphomas are traditionally diagnosed by histology and immunohistochemistry, complemented by molecular methods like fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) to detect the genetic abnormalities that define certain malignancies. To improve both diagnostic accuracy and efficiency, we aimed to implement RNA sequencing (RNAseq) and Whole Exome Sequencing (WES) and flow cytometry into the routine diagnostic workup of pediatric lymph node biopsies. Within a year we analyzed 122 lymph node biopsies of all 110 children referred to our new Dutch National Pediatric Cancer Center because of suspected malignant lymphoma, using a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies. Flow cytometry was performed with fresh tissue directly after biopsy in 83% of specimens (101/122) and allowed rapid diagnosis of mature and immature T- and B-cell non-Hodgkin lymphomas (NHL), enabling early treatment initiation in ill patients. RNAseq was performed on all biopsies of NHL and in a subset of Hodgkin lymphoma (HL) samples. RNAseq detected the vast majority of translocations found by traditional methods. In some cases IgH::MYC translocations were not detected by RNAseq, but for these cases expression analysis showed high MYC expression indicative of the underlying IgH::MYC translocation. In addition, both RNAseq and WES identified additional genetic abnormalities not found by conventional approaches. In several cases WES and/or RNA sequencing resolved remaining diagnostic uncertainty. Machine learning analysis of the RNA sequencing data allowed correct

Cure rates of classical Hodgkin Lymphoma (cHL) in children and young adult patients currently exceed 90%. Nonetheless, survivors are confronted with chronic therapy-related health conditions such as infertility, cardiovascular disease, and high rates of novel second cancers. This calls for development of new targeted and less toxic treatments. cHL is characterized by a low frequency (~0.1-5%) of malignant Hodgkin Reed-Sternberg (HRS) cells, while most of the tissue is composed of nonmalignant immune cells. It is thought that the HRS cells depend on interactions with the tumor microenvironment (TME) for their survival. Indeed, a vast number of interactions between different immune and HRS cells have been reported; however, most of these reports are based on immunohistochemistry or in vitro studies. Here, we systematically characterized the in vivo interactions by applying single-cell RNA sequencing (scRNAseq) to nine primary pediatric and adolescent cHL biopsies and three noncancerous control biopsies of reactive lymph nodes. With scRNAseq, we first sorted live cells to get an unbiased overview of the TME. Then we used a previously published flow cytometry antibody panel to enrich for HRS cells, allowing us to directly assess interactions on a per-tumor basis. Tumor cell identity was confirmed by marker expression as identified by pathology, single-cell copy-number status and the ratio of immunoglobulin kappa/lambda expression. Immune cell identity was determined by canonical marker expression. Using the scRNAseq data, we found that the TME expression profiles in cHL and control biopsies mostly overlap but harbor some differences. First, we identified genes that are consistently overexpressed in HRS cells. These included transcription factors, neural markers, multiple interleukins and other signaling molecules. Second, the extensively described immunosuppressive interactions between HRS, T and NK cells (expressing CTLA-4, TIM-3, and LAG-3) were the strongest and most common interactions that we could identify in HL but not in noncancerous reactive lymph nodes. Third, while the inflammation in the reactive lymph nodes was driven by IFN-g signaling, this pathway was inactive in HL tumors. Other interactions like recruitment of CXCR3+ and CCR4+ T cells, CD47 signaling and interleukin signaling were less pronounced in cHL compared to the controls or were less consistent between tumors. These findings were validated in bulk RNA sequencing of 45 HL tumors. A model arises in which the presence of HRS cells induces inflammation that in most ways resembles lymph node infections. This inflammation and HRS survival are controled by patient-specific interactions between HRS cells and the TME, and by T cell exhaustion, which is universal and the most essential interaction in cHL. Citation Format: Jurrian K. de Kanter, Thanasis Margaritis, Auke Beishuizen, Marijn Scheijde-Vermeulen, Liset Westera, Arianne M. Brandsma, Ruben van Boxtel, Friederike Meyer-Wentrup. Single-cell RNA sequencing reveals that childhood classical Hodgkin Lymphoma resembles normal inflammation except for T cell exhaustion [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A37.